Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa

Abstract

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.

Ultrastructural localization of protein kinase C in human sperm.

“…Signal transduction components have been described in mammalian sperm (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). Recently, we have found PKC and some of its subspecies in human sperm.…”

rab3‐Peptide stimulates exocytosis of the ram sperm acrosome via interaction with cyclic AMP and phospholipase A2 metabolites

“…Throughout the experiments the standard saline incubation medium used consisted of 142 mM NaCI, 2.5 mM KOH, 10 mM glucose and 20 mM HEPES, adjusted to pH 7.55 at 20°C with NaOH [6]; a medium containing 222 mM sucrose in place of the NaC1 was used for washing spermatozoa. Both media also contained 1 mg poly(vinyl) alcohol/ml and 1 mg polyvinylpyrrolidone/ml, and had an osmolality of 305 mOsm/kg.…”

“…Treatment with these agonists, including A23187/Ca 2+, results in a rapid generation of diacylglycerol (DAG) due to the hydrolysis of polyphosphoinositides by phosphoinositidase C [3,[6][7][8] and hydrolysis of phosphatidylcholine by a specific phospholipase C [3,8,9]. No evidence for the participation of the phospholipase D/phosphatidic acid phosphohydrolase pathway in the generation of DAG has been found [3].…”

Bicarbonate/CO2 induces rapid activation of phospholipase A2 and renders boar spermatozoa capable of undergoing acrosomal exocytosis in response to progesterone

“…Phospholipids were separated on 10× 10 cm plastic sheets pretreated by spraying with 1% (w/v) potassium oxalate, activated by heating at I10°C for 10 min, and developed in a two-dimensional thin layer chromatography system [17,19,23]. The plates were air-dried briefly, and the various spots were detected by autoradiography using Fuji RX film.…”